Lipolytic enzyme

ABSTRACT

The present invention relates to novel lipolytic enzymes. More specifically the invention provides novel lipolytic enzymes having the properties of a lipase native to the strain Fusarium culmorum CBS 513.94, or has immunochemical properties identical or partially identical to those of a lipase native to the strain Fusarium culmorum CBS 513.94.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national application ofPCT/DK95/00425 filed Oct. 26, 1995 and claims priority under 35 U.S.C.119 of Danish application 1235/94 filed Oct. 26, 1994, the contents ofwhich are fully incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to novel lipolytic enzymes. Morespecifically the invention provides novel lipolytic enzymes having theproperties of a lipase native to the strain Fusarium culmorum CBS513.94, or has immunochemical properties identical or partiallyidentical to those of a lipase native to the strain Fusarium culmorumCBS 513.94.

BACKGROUND ART

Lipolytic enzymes find multiple industrial applications. Alkalinelipases are of particular interest for use in detergent compositions.

Alkaline lipases of microbial origin have been described, includinglipases obtained from Fusarium. However, lipases obtained from Fusariumculmorum have never been disclosed.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide novel alkalinelipolytic enzymes (EC 3.1.1.3).

Accordingly, in its first aspect, the invention provides lipolyticenzymes having immunochemical properties identical or partiallyidentical to those of a lipase obtained from the strain Fusariumculmorum CBS 513.94.

In its second aspect, the invention provides a process for thepreparation of the lipolytic enzyme, which process comprises cultivationof a lipase producing strain of Fusarium culmorum in a suitable nutrientmedium, containing carbon and nitrogen sources and other inorganicsalts, followed by recovery of the lipolytic enzyme.

In its third aspect, the invention provides a process for thepreparation of a lipolytic enzyme according to any of claims 1-6, whichprocess comprises isolating a DNA fragment encoding the lipolyticenzyme; combining the DNA fragment with an appropriate expression signalin an appropriate plasmid vector; introducing the plasmid vector into anappropriate host either as an autonomously replicating plasmid orintegrated into the chromosome; cultivating the host organism underconditions leading to expression of the lipolytic enzyme; and recoveringof the enzyme from the culture medium.

In further aspects, the invention provides detergent compositions, aswell as a detergent additives, comprising the lipolytic enzyme of theinvention.

Finally, the invention provides a biologically pure culture of thestrain Fusarium culmorum CBS 513.94.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 show the relation between pH and lipase activity of the lipolyticenzyme of the present invention determined at 30° C. in the range offrom pH 6 to pH 10.

DETAILED DISCLOSURE OF THE INVENTION

The present invention provides novel lipolytic enzymes having theproperties of a lipase native to the strain Fusarium culmorum CBS513.94.

The Microorganism

The invention provides lipolytic enzymes derived from a strain of thefungus Fusarium culmorum. Fusarium culmorum is a known species andstrains of Fusarium culmorum have been deposited and are publiclyavailable from depositary institutes, e.g. Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH (DSM), Germany, and American TypeCulture Collection (ATCC), U.S.A.

In a preferred embodiment the invention provides a lipolytic enzymederived from the strain Fusarium culmorum DSM 1094, Fusarium culmorumDSM Fusarium culmorum 62184, Fusarium culmorum DSM 62188, Fusariumculmorum DSM 62191, Fusarium culmorum DSM 62223, Fusarium culmorum ATCC12656, Fusarium culmorum ATCC 15620, Fusarium culmorum ATCC 16430,Fusarium culmorum ATCC 16551, Fusarium culmorum ATCC 26556, Fusariumculmorum ATCC 34910, Fusarium culmorum ATCC 34913, Fusarium culmorumATCC 36017, Fusarium culmorum ATCC 36879, Fusarium culmorum ATCC 36881,Fusarium culmorum ATCC 36886, Fusarium culmorum ATCC 44417, Fusariumculmorum ATCC 46040, Fusarium culmorum ATCC 56088, Fusarium culmorumATCC 56089, Fusarium culmorum ATCC 60275, Fusarium culmorum ATCC 60362,Fusarium culmorum ATCC 62214, Fusarium culmorum ATCC 62215, or Fusariumculmorum ATCC 64075, or a mutant or a variant thereof.

In its most preferred embodiment the invention provides a lipolyticenzyme derived from the strain Fusarium culmorum CBS 513.94, or a muantor a variant thereof. This strain has been deposited according to theBudapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure at CentraalbureauVoor Schimmelcultures (CBS), Oosterstraat 1, Postbus 273, NL-3740 AGBaarn, Netherlands, on 25 Oct. 1994.

In another aspect, the invention provides a biologically pure culture ofthe strain Fusarium culmorum CBS 513.94.

Physico-Chemical Properties

In preferred embodiments, the lipolytic enzyme of the invention may becharacterized by having one or more of the following physico-chemicalproperties.

The enzyme has a pH optimum in the range of from about 7 to about pH 9,more specifically around pH 8, when determined at 30° C. withtributyrine as substrate.

The enzyme has the following N-terminal amino acid sequence (cf. SEQ IDNO:1):

    Ala--Val--Ser--Val--Ser--Thr--Thr--Asp--Phe--Gly--Asn--Phe--Lys--Phe--Tyr--Ile--Gln--His--Gly--Ala--Ala--Ala--Tyr--Xaa--Asn--

The enzyme has a molecular weight of 28.4 kDa, as determined by massspectrometry.

Immunochemical Properties

In another preferred embodiment, the lipolytic enzyme of the inventionis characterized by having having immunochemical properties identical orpartially identical (i.e. at least partially identical) to those of alipase obtained from the strain Fusarium culmorum CBS 513.94.

The immunochemical properties can be determined by immunologicalcross-reaction identity tests. The identity tests can be performed bythe well-known Ouchterlony double immunodiffusion procedure or by tandemcrossed immunoelectrophoresis according to I. M. Roitt; Immunology,Gower Medical Publishing (1985) or N. H. Axelsen; Handbook ofImmunoprecipitation-in-Gel Techniques; Blackwell Scientific Publications(1983), chapters 5 and 14. The terms "immunochemical identity"(antigenic identity) and "partial immunochemical identity" (partialantigenic identity) are described in Axelsen, supra, chapters 5, 19 and20, and in I. M. Roitt, supra, Chapter 6.

Monospecific antiserum for use in immunological tests can be raised,e.g. in rabbits, against the purified lipase of the invention, e.g. asdescribed in Chapter 41 of N. H. Axelsen, supra, or Chapter 23 of N. H.Axelsen et al., A Manual of Quantitative Immunoelectrophoresis,Blackwell Scientific Publications (1973).

Preparation of the Lipolytic Enzyme

The lipolytic enzyme of the invention may be produced by cultivation ofa strain of Fusarium culmorum in a suitable nutrient medium, containingcarbon and nitrogen sources and inorganic salts, followed by recovery ofthe lipase. In a preferred embodiment, the lipase producing strain isthe strain Fusarium culmorum CBS 513.94, or a mutant or a variantthereof.

The lipolytic enzyme may also be obtained by recombinant DNA-technologyby methods known in the art per se, e.g. isolating a DNA fragmentencoding the lipase, combining the DNA fragment with appropriateexpression signal(s) in an appropriate vector, introducing the vector orparts thereof into an appropriate host, either as an autonomouslyreplicating plasmid or integrated into the chromosome, cultivating thehost organism under conditions leading to expression of the lipase, andrecovering the lipase from the culture medium.

In preferred embodiments of the invention, the host organism is ofbacterial origin, preferably a strain of Escherichia coli, or a strainof Bacillus, or a strain of Streptomyces, or of fungal origin,preferably a strain of Aspergillus, a strain of Neurospora, a strain ofFusarium, or a strain of Trichoderma, or a yeast cell, preferably astrain of Saccharomyces, or a strain of Kluyveromyces, or a strain ofHansenula, or a strain of Pichia.

After the cultivation, the lipolytic enzyme may be recovered andpurified from the culture broth by conventional methods, such ashydrophobic chromatography, ion exchange chromatography or combinationsthereof.

Lipolytic Activity

The lipolytic activity may be determined using tributyrine as substrate.This method is based on the hydrolysis of tributyrin by the enzyme, andthe alkali consumption is registered as a function of time.

One Lipase Unit (LU) is defined as the amount of enzyme which, under isstandard conditions (i.e. at 30.0° C.; pH 7.0; and tributyrine assubstrate) liberates 1 μmol titratable butyric acid per minute. GumArabic is used as emulsifier.

A folder AF 95/5 describing this analytical method in more detail isavailable upon request to Novo Nordisk A/S, Denmark, which folder ishereby included by reference.

Detergent Compositions

The lipolytic enzyme of the invention may typically be a component of adetergent composition. As such, it may be included in the detergentcomposition in the form of a non-dusting granulate, a stabilized liquid,or a protected enzyme. Non-dusting granulates may be produced, e.g., asdisclosed in U.S. Pat. Nos. 4,106,991 and 4,661,452 (both to NovoIndustri A/S) and may optionally be coated by methods known in the art.Examples of waxy coating materials are poly(ethylene oxide) products(polyethyleneglycol, PEG) with mean molecular weights of 1000 to 20000;ethoxylated nonylphenols having from 16 to 50 ethylene oxide units;ethoxylated fatty alcohols in which the alcohol contains from 12 to 20carbon atoms and in which there are 15 to 80 ethylene oxide units; fattyalcohols; fatty acids; and mono- and di- and triglycerides of fattyacids. Examples of film-forming coating materials suitable forapplication by fluid bed techniques are given in patent GB 1483591.Liquid enzyme preparations may, for instance, be stabilized by adding apolyol such as propylene glycol, a sugar or sugar alcohol, lactic acidor boric acid according to established methods. Other enzyme stabilizersare well known in the art. Protected enzymes may be prepared accordingto the method disclosed in EP 238,216.

The detergent composition of the invention may be in any convenientform, e.g. as powder, granules, paste or liquid. A liquid detergent maybe aqueous, typically containing up to 70% water and 0-30% organicsolvent, or nonaqueous.

The detergent composition comprises one or more surfactants, each ofwhich may be anionic, nonionic, cationic, or zwitterionic. The detergentwill usually contain 0-50% of anionic surfactant such as linearalkylbenzenesulfonate (LAS), alpha-olefinsulfonate (AOS), alkyl sulfate(fatty alcohol sulfate) (AS), alcohol ethoxysulfate (AEOS or AES),secondary alkanesulfonates (SAS), alpha-sulfo fatty acid methyl esters,alkyl- or alkenylsuccinic acid, or soap. It may also contain 0-40% ofnonionic surfactant such as alcohol ethoxylate (AEO or AE), carboxylatedalcohol ethoxylates, nonylphenol ethoxylate, alkylpolyglycoside,alkyldimethylamine oxide, ethoxylated fatty acid monoethanolamide, fattyacid monoethanolamide, or polyhydroxy alkyl fatty acid amide (e.g. asdescribed in WO 92/06154).

The detergent composition may additionally comprise one or more otherenzymes conventionally used in detergent compositions, such as anamylase, a cutinase, a protease, a cellulase, a peroxidiase, and/or anoxidase.

The detergent may contain 1-65% of a detergent builder or complexingagent such as zeolite, diphosphate, triphosphate, phosphonate, citrate,nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA),diethylenetriaminepentaacetic acid (DTMPA), alkyl- or alkenylsuccinicacid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).The detergent may also be unbuilt, i.e. essentially free of detergentbuilder.

The detergent may comprise one or more polymers. Examples arecarboxymethylcellulose (CMC), poly(vinylpyrrolidone) (PVP),polyethyleneglycol (PEG), poly(vinyl alcohol) (PVA), polycarboxylatessuch as polyacrylates, maleic/acrylic acid copolymers and laurylmethacrylate/acrylic acid copolymers.

The detergent may contain a bleaching system which may comprise a H₂ O₂source such as perborate or percarbonate which may be combined with aperacid-forming bleach activator such as tetraacetylethylenediamine(TAED) or nonanoyloxybenzenesulfonate (NOBS). Alternatively, thebleaching system may comprise peroxyacids of, e.g., the amide, imide, orsulfone type.

The enzymes of the detergent composition of the invention may bestabilized using conventional stabilizing agents, e.g. a polyol such aspropylene glycol or glycerol, a sugar or sugar alcohol, lactic acid,boric acid, or a boric acid derivative such as, e.g., an aromatic borateester, and the composition may be formulated as described in, e.g., WO92/19709 and WO 92/19708.

The detergent may also contain other conventional detergent ingredientssuch as, e.g., fabric conditioners including clays, foam boosters, sudssuppressors, anti-corrosion agents, soil-suspending agents,anti-soil-redeposition agents, dyes, bactericides, optical brighteners,or perfume.

The pH (measured in aqueous solution at use concentration) will usuallybe neutral or alkaline, e.g. in the range of 7-11.

Particular forms of detergent compositions within the scope of theinvention include:

1) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/l comprising

    ______________________________________                                        Linear alkylbenzenesulfonate (calculated as                                                            7-12%                                                acid)                                                                         Alcohol ethoxysulfate (e.g. C.sub.12 -18                                                              1-4%                                                  alcohol, 1-2 EO) or alkyl sulfate (e.g. C.sub.16-18)                          Alcohol ethoxylate (e.g. C.sub.14-15 alcohol,                                                         5-9%                                                  7 EO)                                                                         Sodium carbonate (as Na.sub.2 CO.sub.3)                                                               14-20%                                                Soluble silicate (as Na.sub.2 O,2SiO.sub.2)                                                           2-6%                                                  Zeolite (as NaA1SiO.sub.4)                                                                            15-22%                                                Sodium sulfate (as Na.sub.2 SO.sub.4)                                                                 0-6%                                                  Sodium citrate/citric acid                                                                             0-15%                                                (as C.sub.6 H.sub.5 Na.sub.3 O.sub.7 /C.sub.6 H.sub.8 O.sub.7)                Sodium perborate (as NaBO.sub.3.H.sub.2 O)                                                            11-18%                                                TAED                    2-6%                                                  Carboxymethylcellulose  0-2%                                                  Polymers (e.g. maleic/acrylic acid copolymer,                                                         0-3%                                                  PVP, PEG)                                                                     Enzymes (calculated as pure enzyme                                                                    0.0001-0.1%                                           protein)                                                                      Minor ingredients (e.g. suds suppressors,                                                             0-5%                                                  perfume, optical brightener, photobleach)                                     ______________________________________                                    

2) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/l comprising

    ______________________________________                                        Linear alkylbenzenesulfonate (calculated as                                                           6-11%                                                 acid)                                                                         Alcohol ethoxysulfate (e.g. C.sub.12-18                                                              1-3%                                                   alcohol, 1-2 EO or alkyl sulfate (e.g. C.sub.16-18)                           Alcohol ethoxylate (e.g. C.sub.14-15 alcohol,                                                        5-9%                                                   7 EO)                                                                         Sodium carbonate (as Na.sub.2 CO.sub.3)                                                              15-21%                                                 Soluble silicate (as Na.sub.2 O,2SiO.sub.2)                                                          1-4%                                                   Zeolite (as NaA1SiO.sub.4)                                                                           24-34%                                                 Sodium sulfate (as Na.sub.2 SO.sub.4)                                                                 4-10%                                                 Sodium citrate/citric acid                                                                            0-15%                                                 (as C.sub.6 H.sub.5 Na.sub.3 O.sub.7 /C.sub.6 H.sub.8 O.sub.7)                Carboxymethylcellulose 0-2%                                                   Polymers (e.g. maleic/acrylic acid copolymer,                                                        1-6%                                                   PVP, PEG)                                                                     Enzymes (calculated as pure enzyme                                                                   0.0001-0.1%                                            protein)                                                                      Minor ingredients (e.g. suds suppressors,                                                            0-5%                                                   perfume)                                                                      ______________________________________                                    

3) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/l comprising

    ______________________________________                                        Linear alkylbenzenesulfonate (calculated as                                                          5-9%                                                   acid)                                                                         Alcohol ethoxylate (e.g. C.sub.12-15 alcohol,                                                         7-14%                                                 7 EO)                                                                         Soap as fatty acid (e.g. C.sub.16-22 fatty acid)                                                     1-3%                                                   Sodium carbonate (as Na.sub.2 CO.sub.3)                                                              10-17%                                                 Soluble silicate (as NO,2SiO.sub.2)                                                                  3-9%                                                   Zeolite (as NaA1SiO.sub.4)                                                                           23-33%                                                 Sodium sulfate (as Na.sub.2 SO.sub.4)                                                                0-4%                                                   Sodium perborate (as NaBO.sub.3.H.sub.2 O)                                                            8-16%                                                 TAED                   2-8%                                                   Phosphonate (e.g. EDTMPA)                                                                            0-1%                                                   Carboxymethylcellulose 0-2%                                                   Polymers (e.g. maleic/acrylic acid copolymer,                                                        0-3%                                                   PVP, PEG)                                                                     Enzymes (calculated as pure enzyme                                                                   0.0001-0.1%                                            protein)                                                                      Minor ingredients (e.g. suds suppressors,                                                            0-5%                                                   Perfume, optical brightener)                                                  ______________________________________                                    

4) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/l comprising

    ______________________________________                                        Linear alkylbenzenesulfonate (calculated as                                                           8-12%                                                 acid)                                                                         Alcohol ethoxylate (e.g. C.sub.12-15 alcohol,                                                        10-25%                                                 7 EO)                                                                         Sodium carbonate (as Na.sub.2 CO.sub.3)                                                              14-22%                                                 Soluble silicate (as Na.sub.2 O,2SiO.sub.2)                                                          1-5%                                                   Zeolite (as NaA1SiO.sub.4)                                                                           25-35%                                                 Sodium sulfate (as Na.sub.2 SO.sub.4)                                                                 0-10%                                                 Carboxymethylcellulose 0-2%                                                   Polymers (e.g. maleic/acrylic acid copolymer,                                                        1-3%                                                   PVP, PEG)                                                                     Enzymes (calculated as pure enzyme                                                                   0.0001-0.1%                                            protein)                                                                      Minor ingredients (e.g. suds suppressors,                                                            0-5%                                                   perfume)                                                                      ______________________________________                                    

5) An aqueous liquid detergent composition comprising

    ______________________________________                                        Linear alkylbenzenesulfonate (calculated as                                                          15-21%                                                 acid)                                                                         Alcohol ethoxylate (e.g. C.sub.12-15 alcohol,                                                        12-18%                                                 7 EO or C.sub.12-15 alcohol, 5 EO)                                            Soap as fatty acid (e.g. oleic acid)                                                                  3-13%                                                 Alkenylsuccinic acid (C.sub.12-14)                                                                    0-13%                                                 Aminoethanol            8-18%                                                 Citric acid            2-8%                                                   Phosphonate            0-3%                                                   Polymers (e.g. PVP, PEG)                                                                             0-3%                                                   Borate (as B.sub.4 O.sub.7)                                                                          0-2%                                                   Ethanol                0-3%                                                   Propylene glycol        8-14%                                                 Enzymes (calculated as pure enzyme                                                                   0.0001-0.1%                                            protein)                                                                      Minor ingredients (e.g. dispersants, suds                                                            0-5%                                                   suppressors, perfume, optical brightener)                                     ______________________________________                                    

6) An aqueous structured liquid detergent composition comprising

    ______________________________________                                        Linear alkylbenzenesulfonate (calculated as                                                          15-21%                                                 acid)                                                                         Alcohol ethoxylate (e.g. C.sub.12-15 alcohol,                                                        3-9%                                                   7 EO, or C.sub.12-15 alcohol, 5 EO)                                           Soap as fatty acid (e.g. oleic acid)                                                                  3-10%                                                 Zeolite (as NaA1SiO.sub.4)                                                                           14-22%                                                 Potassium citrate       9-18%                                                 Borate (as B.sub.4 O.sub.7)                                                                          0-2%                                                   Carboxymethylcellulose 0-2%                                                   Polymers (e.g. PEG, PVP)                                                                             0-3%                                                   Anchoring polymers such as, e.g., lauryl                                                             0-3%                                                   methacrylate/acrylic acid copolymer; molar                                    ratio 25:1; MW 3800                                                           Glycerol               0-5%                                                   Enzymes (calculated as pure enzyme protein)                                                          0.0001-0.1%                                            Minor ingredients (e.g. dispersants, suds                                                            0-5%                                                   suppressors, perfume, optical brighteners)                                    ______________________________________                                    

7) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/l comprising

    ______________________________________                                        Fatty alcohol sulfate   5-10%                                                 Ethoxylated fatty acid monoethanolamide                                                              3-9%                                                   Soap as fatty acid     0-3%                                                   Sodium carbonate (as Na.sub.2 CO.sub.3)                                                               5-10%                                                 Soluble silicate (as Na.sub.2 O,2SiO.sub.2)                                                          1-4%                                                   Zeolite (as NaA1SiO.sub.4)                                                                           20-40%                                                 Sodium sulfate (as Na.sub.2 SO.sub.4)                                                                2-8%                                                   Sodium perborate (as NaBO.sub.3.H.sub.2 O)                                                           12-18%                                                 TAED                   2-7%                                                   Polymers (e.g. maleic/acrylic acid copolymer,                                                        1-5%                                                   PEG)                                                                          Enzymes (calculated as pure enzyme                                                                   0.0001-0.1%                                            protein)                                                                      Minor ingredients (e.g. optical brightener, suds                                                     0-5%                                                   suppressors, perfume)                                                         ______________________________________                                    

8) A detergent composition formulated as a granulate comprising

    ______________________________________                                        Linear alkylbenzenesulfonate (calculated as                                                           8-14%                                                 acid)                                                                         Ethoxylated fatty acid monoethanolamide                                                               5-11%                                                 Soap as fatty acid     0-3%                                                   Sodium carbonate (as Na.sub.2 CO.sub.3)                                                               4-10%                                                 Soluble silicate (as Na.sub.2 O,2SiO.sub.2)                                                          1-4%                                                   Zeolite (as NaA1SiO.sub.4)                                                                           30-50%                                                 Sodium sulfate (as Na.sub.2 SO.sub.4)                                                                 3-11%                                                 Sodium citrate (as C.sub.6 H.sub.5 Na.sub.3 O.sub.7)                                                  5-12%                                                 Polymers (e.g. PVP, maleic/acrylic acid                                                              1-5%                                                   copolymer, PEG)                                                               Enzymes (calculated as pure enzyme                                                                   0.0001-0.1%                                            protein)                                                                      Minor ingredients (e.g. suds suppressors,                                                            0-5%                                                   perfume)                                                                      ______________________________________                                    

9) A detergent composition formulated as a granulate comprising

    ______________________________________                                        Linear alkylbenzenesulfonate (calculated as                                                           6-12%                                                 acid)                                                                         Nonionic surfactant    1-4%                                                   Soap as fatty acid     2-6%                                                   Sodium carbonate (as Na.sub.2 CO.sub.3)                                                              14-22%                                                 Zeolite (as NaA1SiO.sub.4)                                                                           18-32%                                                 Sodium sulfate (as Na.sub.2 SO.sub.4)                                                                 5-20%                                                 Sodium citrate (as C.sub.6 H.sub.5 Na.sub.3 O.sub.7)                                                 3-8%                                                   Sodium perborate (as NaBO.sub.3.H.sub.2 O)                                                           4-9%                                                   Bleach activator (e.g. NOBS or TAED)                                                                 1-5%                                                   Carboxymethylcellulose 0-2%                                                   Polymers (e.g. polycarboxylate or PEG)                                                               1-5%                                                   Enzymes (calculated as pure enzyme                                                                   0.0001-0.1%                                            protein)                                                                      Minor ingredients (e.g. optical brightener                                                           0-5%                                                   perfume)                                                                      ______________________________________                                    

10) An aqueous liquid detergent composition comprising

    ______________________________________                                        Linear alkylbenzenesulfonate (calculated as                                                          15-23%                                                 acid)                                                                         Alcohol ethoxysulfate (e.g. C.sub.12-15                                                               8-15%                                                 alcohol, 2-3 EO)                                                              Alcohol ethoxylate (e.g. C.sub.12-15 alcohol, 7 EO,                                                  3-9%                                                   or C.sub.12-15 alcohol, 5 EO)                                                 Soap as fatty acid (e.g. lauric acid)                                                                0-3%                                                   Aminoethanol           1-5%                                                   Sodium citrate          5-10%                                                 Hydrotrope (e.g. sodium toluensulfonate)                                                             2-6%                                                   Borate (as B.sub.4 O.sub.7)                                                                          0-2%                                                   Carboxymethylcellulose 0-1%                                                   Ethanol                1-3%                                                   Propylene glycol       2-5%                                                   Enzymes (calculated as pure enzyme protein)                                                          0.0001-0.1%                                            Minor ingredients (e.g. polymers, dispersants,                                                       0-5%                                                   perfume, optical brighteners)                                                 ______________________________________                                    

11) An aqueous liquid detergent composition comprising

    ______________________________________                                        Linear alkylbenzenesulfonate (calculated as                                                          20-32%                                                 acid)                                                                         Alcohol ethoxylate (e.g. C.sub.12-15 alcohol,                                                         6-12%                                                 7 EO, or C.sub.12-15 alcohol, 5 EO)                                           Aminoethanol           2-6%                                                   Citric acid             8-14%                                                 Borate (as B.sub.4 O.sub.7)                                                                          1-3%                                                   Polymer (e.g. maleic/acrylic acid copolymer,                                                         0-3%                                                   anchoring polymer such as, e.g., lauryl                                       methacrylate/acrylic acid copolymer)                                          Glycerol               3-8%                                                   Enzymes (calculated as pure enzyme                                                                   0.0001-0.1%                                            protein)                                                                      Minor ingredients (e.g. hydrotropes,                                                                 0-5%                                                   dispersants, perfume, optical brighteners)                                    ______________________________________                                    

12) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/l comprising

    ______________________________________                                        Anionic surfactant (linear alkylbenzene-                                                             25-40%                                                 sulfonate, alkyl sulfate, alpha-olefinsulfonate,                              alpha-sulfo fatty acid methyl esters,                                         alkanesultonates, soap)                                                       Nonionic surfactant (e.g. alcohol                                                                     1-10%                                                 ethoxylate)                                                                   Sodium carbonate (as Na.sub.2 CO.sub.3)                                                               8-25%                                                 Soluble silicates (as Na.sub.2 O, 2SiO.sub.2)                                                         5-15%                                                 Sodium sulfate (as Na.sub.2 SO.sub.4)                                                                0-5%                                                   Zeolite (as NaA1SiO.sub.4)                                                                           15-28%                                                 Sodium perborate (as NaBO.sub.3.4H.sub.2 O)                                                           0-20%                                                 Bleach activator (TAED or NOBS)                                                                      0-5%                                                   Enzymes (calculated as pure enzyme                                                                   0.0001-0.1%                                            protein)                                                                      Minor ingredients (e.g. perfume, optical                                                             0-3%                                                   brighteners)                                                                  ______________________________________                                    

13) Detergent formulations as described in 1)-12) wherein all or part ofthe linear alkylbenzenesulfonate is replaced by (C₁₂ -C₁₈) alkylsulfate.

14) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/l comprising

    ______________________________________                                        (C.sub.12 --C.sub.18) alkyl sulfate                                                                   9-15%                                                 Alcohol ethoxylate     3-6%                                                   Polyhydroxy alkyl fatty acid amide                                                                   1-5%                                                   Zeolite (as NaA1SiO.sub.4)                                                                           10-20%                                                 Layered disilicate (e.g. SK56 from Hoechst)                                                          10-20%                                                 Sodium carbonate (as Na.sub.2 CO.sub.3)                                                               3-12%                                                 Soluble silicate (as Na.sub.2 O,2SiO.sub.2)                                                          0-6%                                                   Sodium citrate         4-8%                                                   Sodium percarbonate    13-22%                                                 TAED                   3-8%                                                   Polymers (e.g. polycarboxylate and PVP =                                                             0-5%                                                   Enzymes (calculated as pure enzyme                                                                   0.0001-0.1%                                            protein)                                                                      Minor ingredients (e.g. optical brightener,                                                          0-5%                                                   photo bleach, perfume, suds suppressors)                                      ______________________________________                                    

15) A detergent composition formulated as a granulate having a bulkdensity of at least 600 g/l comprising

    ______________________________________                                        (C.sub.12 --C.sub.18) alkyl sulfate                                                                 4-8%                                                    Alcohol ethoxylate    11-15%                                                  Soap                  1-4%                                                    Zeolite MAP or zeolite A                                                                            35-45%                                                  Sodium carbonate (as Na.sub.2 CO.sub.3)                                                             2-8%                                                    Soluble silicate (as Na.sub.2 O,2SiO.sub.2)                                                         0-4%                                                    Sodium percarbonate   13-22%                                                  TAED                  1-8%                                                    Carboxymethyl cellulose                                                                             0-3%                                                    Polymers (e.g. polycarboxylates and PVP)                                                            0-3%                                                    Enzymes (calculated as pure enzyme                                                                  0.0001-0.1%                                             protein)                                                                      Minor ingredients (e.g. optical brightener,                                                         0-3%                                                    phosphonate, perfume)                                                         ______________________________________                                    

16) Detergent formulations as described in 1)-15) which contain astabilized or encapsulated peracid, either as an additional component oras a substitute for already specified bleach systems.

17) Detergent compositions as described in 1), 3), 7), 9) and 12)wherein perborate is replaced by percarbonate.

18) Detergent compositions as described in 1), 3), 7), 9), 12), 14) and15) which additionally contain a manganese catalyst. The manganesecatalyst may, e.g., be one of the compounds described in "Efficientmanganese catalysts for low-temperature bleaching", Nature 369, 1994,pp. 637-639.

19) Detergent composition formulated as a nonaqueous detergent liquidcomprising a liquid nonionic surfactant such as, e.g., linearalkoxylated primary alcohol, a builder system (e.g. phosphate), enzymeand alkali. The detergent may also comprise anionic surfactant and/or ableach system.

The lipolytic enzyme of the invention may be incorporated inconcentrations conventionally employed in detergents. It is at presentcontemplated that, in the detergent composition of the invention, thelipase may be added in an amount corresponding to 0.001-100 mg of lipaseper liter of wash liquor.

EXAMPLES

The invention is further illustrated with reference to the followingexamples which are not intended to be in any way limiting to the scopeof the invention as claimed.

Example 1 Cultivation Example

Seed cultures of the strain Fusarium culmorum CBS 513.94 were producedin 500 ml shakeflasks containing 100 ml of the following composition:

Corn steep liquer (dried) 12 g/l

Glucose 24 g/l

To each flask is added 0.5 g CaCO₃ and 0.5 ml of oil.

pH is adjusted to 5.5 before autoclavation.

After 3 days at 26° C. and 250 rpm, 5 ml of each of the seed cultureswere inoculated in shakeflasks containing 100 ml of the followingmedium:

Pepton, Difco 0118 6 g/l

Pepticase, Sheffield Products 4 g/l

Yeast extract, Difco 0127 3 g/l

Meat extract, Difco 0126 1.5 g/l

Dextrose, Roquette 101-0441 1 g/l

Olive oil, Sigma 10 g/l

pH is adjusted to 7.3-7.4 before autoclavation.

Cultivation took place for 9 days at 26° C. and 250 rpm. The broths werecentrifuged and the supernatants purified on a hydrophobic matrix (TSKgel Butyl-ToyoPearl 650 C column, available from Tosoh Corporation,Japan), and applied for further studies.

Example 2 Characterization Example

pH Optimum

The supernatant obtained according to Example 1 was subjected to the LUmethod for determining lipase activity described above, and the relationbetween pH and lipase activity of the lipolytic enzyme of the inventionwas determined at 30° C. in the range of from pH 6 to pH 10.

The results of this characterization is presented in FIG. 1. Thelipolytic enzyme has its pH optimum in the range of from about pH 7 toabout pH 9, more specifically around pH 8.

Molecular Weight Determination

Mass spectrometry was done using matrix assisted laser desorptionionisation time-of flight (MALDI-TOF) mass spectrometry in a VGAnalytical TofSpec. For mass spectrometry, 2 μl of sample obtainedaccording to Example 1 were mixed with 2 μl saturated matrix solution(α-cyano-4-hydroxycinnamic acid in 0.1% TFA:acetonitrile (70:30)), and 2μl of the mixture spotted onto the target plate. Before introductioninto the mass spectrometer the solvent was removed by evaporation. Thesample was desorbed and ionised by 4 ns laser pulses (337 nm) atthreshold laser power and accelerated into the field-free flight tube byan accelerating voltage of 25 kV. Ions were detected by a micro channelplate set at 1850 V. The spectra were calibrated externally withproteins of known mass.

A mass of 28.4 kDa was determined.

N-terminal Amino Acid Sequence

Using standard methods for obtaining and sequencing peptides Findlay &Geisow (Eds.) (1989); Protein sequencing--a practical approach; IRLPress!, the following 25 N-terminal amino acid residues of the lipolyticenzyme have been identified, as presented by SEQ ID NO:1 (where Xaadesignates an unknown amino acid residue):

    Ala--Val--Ser--Val--Ser--Thr--Thr--Asp--Phe--Gly--Asn--Phe--Lys--Phe--Tyr--Ile--Gln--His--Gly--Ala--Ala--Ala--Tyr--Xaa--Asn--

Example 3 Lipolytic Activity

Using a monolayer equipment (KSV-5000, KSV Instruments, Finland) it hasbeen demonstrated that the lipolytic enzyme from Fusarium culmorum hasconsiderably increased activity towards dicaprin in presence of longchained alcoholethoxylates.

A mixed monolayer in a well defined overall composition, made of adiglyceride substrate and a monocomponent alcoholethoxylate (AEO:Heptaethylene glycol monooctadecyl ether) is spread on an aqueoussubphase (10 mM Glycine, pH 10.0, 0.1 mM EDTA, 25° C.). The surfacepressure is adjusted to the desired value, and a well-defined amount ofenzyme (10 LU; lipase units as defined above) is injected into thesubphase. Lipolytic action is manifested through the speed of a mobilebarrier compressing the monolayer in order to maintain constant surfacepressure as insoluble substrate molecules are hydrolysed into more watersoluble reaction products. Using this assay, lipolytic enzymes arediscriminated by a parameter β indicating the final area-fraction ofsubstrate (dicaprin) left unhydrolysed by the enzyme as lipolyticactivity stops.

In this way, the lipase of the invention was compared to an Aspergilluslipase conventionally used in detergents (Lipolase™, available from NovoNordisk A/S, Denmark). The results are presented in Table 1, below.

                  TABLE 1                                                         ______________________________________                                        Improved tolerance of lipolytic enzyme from                                   Fusarium culmorum compared to Lipolase ™.                                                  β(30 mN/m)*                                              ______________________________________                                        Lipolase ™     57%                                                         Fusarium culmorum lipase                                                                        25%                                                         ______________________________________                                         *Surface pressure employed.                                              

These results show that when compared to Lipolase™, the lipolytic enzymeobtained from Fusarium culmorum is considerably more efficient whenalcoholethoxylates are present in the substrate phase.

Example 4 Substrate Affinity

A procedure has been developed aiming at a simple comparison of theability of lipolytic enzymes to accumulate on/in a substrate phase(olive oil) at alkaline pH (pH 9.0) and presence of the non-ionicsurfactant Dobanol 25-7 (2500 ppm) in the aqueous phase.

Procedure

1. Two identical buffer solutions (5 ml) are prepared in 20 ml sealablevials, ("Sample" (s) and "Reference" (r)).

2. Enzyme is added into "Sample" and "Reference" and the lipaseconcentration is determined (X LU/ml).

3. Olive oil is added onto the "Sample" and both lipase solutions areshaken vigorously. Incubation at 4° C. over night.

4. Remaining lipase concentration in the aqueous phases is determinedafter incubation (Y₁ LU/mi; i=r,s).

Summary of Incubation Conditions

Buffer 100 mM Glycine (5 ml).

pH 9.0.

Substrate Olive oil (5 ml).

Temperature 4° C.

Lipase activity 5-10 LU/ml.

Incubation Over night (24-26 hours).

Evaluation of Data

The result is calculated by comparing the activity-loss upon incubationin the aqueous phase in contact with olive oil to the activity-loss inthe aqueous phase in absence of olive oil:

    α=Y.sub.s /Y.sub.f (see above)

The results are presented in Table 2, below.

                  TABLE 2                                                         ______________________________________                                        Substrate Affinity                                                            Lipolytic Enzyme   α(%)                                                 ______________________________________                                        Lipolase ™      99%                                                        Fusarium culmorum lipase                                                                         99%                                                        ______________________________________                                    

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 1                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AlaValSerValSerThrThrAspPheGlyAsnPheLysPheTyrIle                              151015                                                                        GlnHisGlyAlaAlaAlaTyrXaaAsn                                                   2025                                                                          __________________________________________________________________________

We claim:
 1. A lipolytic enzyme having immunochemical properties atleast partially identical to those of a lipase obtained from the strainFusarium culmorum CBS 513.94, which enzyme has the N-terminal amino acidsequence shown in SEQ ID NO: 1, in which Xaa denotes any amino acidresidue.
 2. The lipolytic enzyme of claim 1, which is obtained from astrain of Fusarium culmorum.
 3. The lipolytic enzyme of claim 1, whichis obtained from the strain Fusarium culmorum CBS 513.94, or a mutant ora variant thereof.
 4. The lipolytic enzyme of claim 1, which has a pHoptimum in the range of from about pH 7 to about pH 9, when determinedat 30° C. with tributyrine as substrate.
 5. The lipolytic enzyme ofclaim 1, which has a molecular weight of 28.4 kDa, as measured bySDS-PAGE.
 6. A process for the preparation of the lipolytic enzyme ofclaim 1, which process comprises cultivation of a lipase producingstrain of Fusarium culmorum in a suitable nutrient medium, containingcarbon and nitrogen sources and other inorganic salts, followed byrecovery of the lipolytic enzyme.
 7. A detergent composition comprisingthe lipolytic enzyme of claim
 1. 8. The detergent composition of claim7, which further comprises at least one other enzyme.
 9. The detergentcomposition of claim 8, wherein the other enzyme is a protease, anamylase, a cellulase, an oxidase or a peroxidase.
 10. A detergentadditive comprising the lipolytic enzyme of claim 1, provided in theform of a granulate, a liquid, a slurry or a protected enzyme.
 11. Thedetergent additive of claim 10, wherein the granulate is a non-dustinggranulate and the liquid is a stabilized liquid.